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New evidence appearing in today’s Nature implicates the ApoE4 allele, the primary genetic risk factor for late-onset Alzheimer’s disease, as a prime culprit in damaging brain blood vessels. Although scientists suspected that ApoE4 worked some mischief at the blood-brain barrier (BBB), the mechanism was unknown. Now, researchers led by Berislav Zlokovic, previously at the University of Rochester, New York, and now at the University of Southern California, Los Angeles, describe a detailed inflammatory pathway through which human ApoE4 triggers BBB breakdown in transgenic mice. This breakdown causes toxic serum proteins to accumulate in the brain and provokes neuronal degeneration, the authors report. Notably, this occurs in the absence of any Aβ. Zlokovic and colleagues were able to restore the BBB and improve the neuronal health of the mice through genetic and pharmacological manipulations of the pathway, suggesting this mechanism could be a therapeutic target in ApoE4 carriers, who make up the majority of sporadic AD cases. However, it remains to be seen whether the results will translate to humans, and whether the pathway will be amenable to drug development. First author Robert Bell at the University of Rochester previously presented some of this research at the 2010 Society for Neuroscience annual conference in San Diego, California (see ARF related news story).

Other scientists contacted by Alzforum expressed enthusiasm for the findings. “The paper is a tour de force in pinning down a role for ApoE in vascular protection,” said Cheryl Wellington at the University of British Columbia, Vancouver, Canada. “This is an important paper, because it describes a very early event in AD pathogenesis that could precede a lot of other downstream things.”

Researchers have struggled to nail down precisely how ApoE4 inflates AD risk because the protein acts on so many cellular processes (see ARF related news story). In particular, ApoE4 promotes amyloid-β deposition, giving it a direct role in AD pathology (see ARF related news story). How does Aβ, which is present in human AD, relate to ApoE’s actions at the BBB? Zlokovic told Alzforum he favors a two-hit hypothesis. He believes ApoE4 first damages the cerebrovasculature, kicking off a cascade of brain damage, then, as a second hit, amplifies Aβ deposition (see Zlokovic, 2011). Other scientists agreed that the BBB mechanism is probably an important contributor to brain damage, but is unlikely to explain all of the AD risk conferred by ApoE4, and may act in tandem with Aβ-dependent pathways. “Cerebrovascular degeneration in concert with Aβ [deposition] could have a synergistic effect on cognition,” suggested Donna Wilcock at the University of Kentucky, Lexington.

To study the vascular role of ApoE, Bell and colleagues used mice in which the endogenous mouse protein was replaced with human ApoE2, 3, or 4, as well as ApoE knockout animals. In two-week-old mice with ApoE4 or no ApoE, cerebral blood vessels leaked profusely, capillary length declined, and cerebral blood flow dropped. These changes grew worse with age. Intriguingly, levels of the proinflammatory cytokine, cyclophilin A (CypA), which has been shown to damage blood vessels (see Satoh et al., 2009; Jin et al., 2004), jumped fivefold in these animals compared to ApoE2 and ApoE3 mice. The CypA explosion occurred in pericytes, a type of cell that wraps around small blood vessels. When the authors crossed ApoE4 and knockout animals with mice lacking CypA, the BBB remained intact. Feeding the mice cyclosporine A, a CypA inhibitor, also tightened up the BBB.

The authors then dissected how ApoE4 induces CypA. They found that ApoE, which is predominantly made by astrocytes in the brain, binds to low-density lipoprotein receptor-related protein 1 (LRP1) on pericytes. The ApoE4 allele, however, fails to bind the receptor, as shown by proximity ligation assay, a highly sensitive type of immunoassay (see Fredriksson et al., 2002; Söderberg et al., 2006). In ApoE4 or ApoE knockout mice, CypA synthesis goes wild. The cytokine then activates pericyte nuclear factor κB (NF-κB), which translocates to the nucleus and pumps up production of matrix metalloproteinase 9 (MMP9). This proteinase chews up capillary basement membrane and tight junction proteins, effectively punching holes in the blood-brain barrier. Interfering with any step in this pathway, by pharmacological inhibitors, short interfering RNA, or genetic deletion, restored BBB function, the authors report.

Furthermore, the brains of ApoE4 and knockout mice accumulated serum proteins such as fibrin, thrombin, and hemosiderin, which can poison neurons (see Grammas, 2011; Paul et al., 2007). Bell and colleagues showed that, by four months of age, ApoE4 mice had less neuronal activity and were losing neurites and synaptic proteins. Inhibiting the CypA-MMP9 pathway partially reversed this neurodegeneration, improving neuron structure and function. In future work, Zlokovic said he will test behavior and information processing in these mice to see if the neuronal losses correlate with cognitive problems.

One big question is whether these findings relate to humans. Zlokovic plans to examine cerebrospinal fluid from AD patients to see if the main markers of this inflammatory pathway, CypA and MMP9, are elevated in people with the ApoE4 allele. In collaboration with colleagues at the University of Southern California, he is also developing methods using MRI to look at BBB health in AD patients. The task is challenging, because small flaws in capillaries typically do not show up on MRIs, he noted.

Neurodegenerative conditions such as AD frequently go hand-in-hand with BBB disruption (see, e.g., Farrall and Wardlaw, 2009; Dickstein et al., 2010), and vascular flaws appear to be more pronounced in people carrying the ApoE4 allele (see Salloway et al., 2002). People with the ApoE4 allele are known to have higher levels of cerebral amyloid angiopathy (CAA) and be more susceptible to microhemorrhages compared to non-carriers, which has caused problems for this group in immunotherapy trials, Wilcock pointed out (see, e.g., ARF related news story). Wilcock recently showed that giving immunotherapy to mice activates the MMP9 pathway, which may help explain some of the vascular damage seen in trials (see Wilcock et al., 2011). Zlokovic’s study now highlights why ApoE4 carriers may be particularly susceptible to this mechanism. It also holds out hope that “Maybe we now have a targetable pathway in ApoE4s,” Wilcock said. “I think all of this is going to start pointing us toward a more personalized therapeutic approach, as opposed to a one-size-fits-all.”

The ApoE4 allele is also a risk factor for other neurodegenerative conditions, such as Parkinson’s and multiple sclerosis (see ARF related news story). Having an E4 allele worsens a person’s outcome after ischemic or traumatic brain injury, noted Yadong Huang at the Gladstone Institute for Neurological Disease, San Francisco, California (see Mayeux et al., 1995 and ARF related news story). Huang believes the new findings have relevance for these conditions, too. “Whenever you have trauma or brain injury, cerebrovascular integrity is critical,” he said.

While the new data suggest that inhibiting the CypA-MMP9 pathway could reverse vascular problems and perhaps prevent brain damage in ApoE4 carriers, the route from an academic result to a usable drug is a long and arduous one, cautioned Ryan Watts at Genentech, South San Francisco, California. Because most chemical inhibitors are “dirty,” hitting many targets, researchers need to perform careful pharmacodynamic and pharmacokinetic studies over a range of doses to be sure a drug is selectively inhibiting the desired target, he said. Also, molecules such as MMP9 and CypA act on many pathways and have beneficial effects as well, which means that inhibiting them long term could have undesirable side effects. “Although promising, substantially more work will be necessary to determine if these pathways/targets proposed in this manuscript are suitable for the treatment of Alzheimer's and other diseases associated with ApoE4,” Watts wrote (see full comment below).—Madolyn Bowman Rogers

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This is a very interesting manuscript that is noteworthy for its proposed mechanism linking ApoE and cerebrovascular dysfunction. The authors use a combination of genetic and pharmacological manipulations to propose a link between ApoE, cyclophilin A, and MMP9 activation. Although promising, substantially more work will be necessary to determine if the pathways/targets proposed in this manuscript are suitable for the treatment of Alzheimer's and other diseases associated with ApoE4. Below are several points worthy of further consideration:

1. The work offers yet another model by which ApoE may be mediating a general neurotoxic outcome in the brain (to be added to the many others already in the literature). As ApoE is linked to several degenerative diseases, and recovery after stroke, a general mechanism as proposed by Zlokovic and colleagues is reasonable; however, there is a wide range of previous work claiming different "general" mechanisms. My fear is that ApoE4 is pleiotropic, affecting a number of cell biological mechanisms; thus, pinpointing a specific cellular mechanism may prove elusive. Nevertheless, the authors make a concerted effort to establish a molecular mechanism driving blood-brain barrier disruption, namely, the activation of MMP9 via cyclophilin A, dismantling of the basement membrane, and downregulation of proteins regulating endothelial tight junctions.

2. Although the genetic manipulations look compelling, caution is necessary when interpreting results from pharmacological manipulations. There is no extensive pharmacokinetics/pharmacodynamics to fully assure the reader that these molecules (cyclosporine, PDTC, and SB-3CT) are specifically acting via the mechanisms proposed.

3. The model proposed by Zlokovic and colleagues does not account for the most validated observation related to Alzheimer's and ApoE4, namely, that ApoE4 increases the risk of developing amyloid plaques. Furthermore, it has been proposed that ApoE4 carriers show a reduction in Aβ efflux. This being said, it is not unreasonable to assume that ApoE is modulating multiple areas of biology as discussed above.

This paper represents a natural progression for the group that has clarified the effect of different isoforms of ApoE on the transporters that clear Aβ from the brain. Here, the group has made significant contributions in demonstrating how ApoE exerts its effect on the blood-brain barrier. The authors demonstrated first that the absence of ApoE or the presence of human ApoE4 in mice results in a leaky blood-brain barrier, associated with decreased levels of proteins expressed at the tight junctions, and decreased levels of collagen IV. Collagen IV is a glycoprotein present at the basement membranes, and it prevents the formation of Aβ fibrils. Apart from their clearance across the endothelium into the blood, solutes and Aβ are eliminated by perivascular drainage along cerebrovascular basement membranes (Hawkes et al., 2011) .

The authors then demonstrate that ApoE4 is associated with high expression of cyclophilin A (CypA) in pericytes and increased expression of matrix metalloproteinase 9 (MMP9). A series of very elegant in-vivo experiments, coupled with pharmacological and genetic manipulation, demonstrates that CypA, nuclear factor κB, and MMP9 are responsible for the breakdown in the BBB observed in the presence of ApoE4. Furthermore, the study demonstrates that the endothelium lipoprotein receptor responsible for clearance of Aβ is also present on pericytes. Isoforms of ApoE regulate the expression and function of lipoprotein receptor on pericytes. ApoE3 binds with high affinity to LRP1, whereas ApoE4 does not bind to pericyte LRP1, a result very similar to that observed in previous studies of the interactions of ApoE and LRP1 at the vascular level.

Recently, this group led by Berislav Zlokovic has clarified many of the physiological roles of pericytes in maintaining the integrity of the neurovascular unit (Winkler et al., 2011). The present study, through a series of important findings about how pericytes interact with ApoE and influence the integrity of the blood-brain barrier, is a major step in clarifying the factors behind the pathogenesis of neurodegenerative disorders. Pericytes may provide the motive force for the drainage of solutes from the extracellular spaces along vascular basement membranes. Defects in the function of pericytes may be associated with a failure of elimination of Aβ by lipoprotein receptors as well as by perivascular drainage. It is possible that targeting the activity of pericytes may become a therapeutic strategy in the treatment of neurodegenerative diseases.

Fascinating paper! I am somewhat hesitant to extrapolate its relevance directly to humans; intuitively the effects seem too large for this. But mechanistically, the findings are concordant with Nishitsuji et al. This will require confirmation, of course, but the idea and the plausible mechanism definitely warrant detailed scrutiny and extension of these studies by other labs. For instance, Boucher et al. showed that loss of LRP1 in vascular smooth muscle cells results in increased activation of MMP2 and MMP9, which fits well with the results reported here by Bell et al.

What I find further tantalizing is the link it offers to cerebral amyloid angiopathy, which occurs so frequently in ApoE4 carriers. I wonder how exactly these mechanisms might be connected. On the other hand, if human ApoE4 carriers were suffering from such a large degree of blood-brain barrier (BBB) leakage, would one not expect this to manifest itself clinically in a more prominent manner? Perhaps the effect in humans is smaller than in the mouse? On the other hand, an increased incidence of glomerular nephropathy has also been reported to be associated with ApoE4, raising the possibility that the ApoE4 effect at the BBB may extend to the related mesangial cells in the kidney glomerulus.

ApoE, Microvascular Injury, and Blood-Brain Barrier Compromise in Sporadic (Late-Onset) Alzheimer’s Disease: A Shining New Light for Therapeutic Intervention
Alzheimer’s disease (AD) is a genetically diverse spectrum of disorders that includes both familial and sporadic forms (1). The familial forms of the disease are seen in less than 10 percent of cases, and are associated with mutations on chromosomes 21 (amyloid precursor protein) (2-4), 14 (presenilin I) (5-7), and 1 (presenilin II) (8-9). Patients generally present with symptoms of cognitive impairment at an early age, have a rapidly progressive course, and exhibit severe pathologic alterations in their brains. Patients with the more common late-onset sporadic form of the disease (90 percent) are likely to be homozygous for the ApoE4 gene on chromosome 19, which codes for the high-density lipoprotein ApoE4 (10). Such patients typically exhibit symptoms of cognitive impairment later in life, have a more slowly progressive clinical course, and a variable degree of brain AD pathology. Despite the unequivocal association between ApoE4 and late-onset sporadic AD, the mechanism(s) through which ApoE4 contributes to the pathogenesis of sporadic AD remain(s) elusive.

Numerous brain imaging studies by SPECT, CT, PET, and MRI have documented a preferential decrease in cerebral blood flow to brain areas affected by AD, as well as an increase in small vessel disease in Alzheimer's patients (11-18). Microvascular disease is a common finding at autopsy in the brains of elderly patients, and significant microvascular pathology has been extensively described in AD (19-25). Various components of the fragmented vascular basement membrane are found within senile (neuritic) plaques, raising the question of whether plaque formation and microvascular pathology are somehow closely linked (26-30). Previous studies by our group and others have documented that agrin, the major heparan-sulfate proteoglycan component of the cerebral capillary basement membrane, becomes fragmented in sporadic AD, compromising microvascular structural integrity (31-34). We have also demonstrated that this structural damage is greater in AD patients with the ApoE4 genotype, and correlates with the appearance of serum-derived proteins in the brain, presumably due to a defective blood-brain barrier (35-36).

Thus far, evidence supporting a derangement in blood-brain barrier integrity in AD has been derived from clinical studies using the CSF/serum protein ratios of albumin, haptoglobin, and IgG. These studies can be roughly divided into two groups: those finding no evidence of a blood-brain barrier defect in AD (37-40), and those concluding that there is a significant compromise in blood-brain barrier integrity (41-45). Problems with experimental design may account for some of these discrepancies. Sample sizes were often limited to a very small number of patients. Most of the earlier studies failed to consider the severity of AD as a significant variable in their analyses, combining patients with both early and advanced disease into the same AD cohort. Clinical criteria for the diagnosis of AD were often vaguely defined. A trend for improvement in study design is evident in the three most recent studies, which have all concluded that blood-brain barrier integrity is clearly compromised in AD patients (43-45).

This landmark paper by Bell et al. demonstrates, through an elegant series of experiments in genetically altered mice, that expression of human ApoE4 and lack of murine ApoE leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes (46). This then leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. The potential therapeutic relevance of these animal model investigations is strongly supported by prior studies using postmortem brain tissue from Alzheimer's patients, generously provided by their families.

Aging and brain trauma in human patients may both impair the BBB (perhaps synergistically) through the exact mechanisms described in this exciting report, setting into motion a cascade of pathologic processes that destabilize brain fluid homeostasis and lead to cognitive decline. Information gained from these experiments may lead to earlier identification and therapeutic intervention. Pharmacologic and epigenetic manipulations, related to preserving the neurovascular unit and BBB, clearly represent an exciting new approach for reducing the onset and progression of dementia in sporadic AD patients.

This paper by Bell and colleagues reports exciting findings that suggest novel mechanisms underlying the role of ApoE genotype in neurodegeneration. They implicate ApoE4 in the breakdown of blood-brain barrier (BBB) integrity, an effect that is mediated by cyclophilin A. The compromised BBB appears to facilitate accumulation of blood-derived neurotoxic proteins, including fibrin, hemosiderin, and thrombin in ApoE4 mice. The authors delineate the temporal course of these changes and provide evidence that vascular dysfunction as reflected in disruption of the BBB precedes neuronal dysfunction in ApoE-negative and ApoE4 mice. These findings provide novel insights into the role of ApoE genotype in provoking neuronal dysfunction/synaptic failure. While extrapolating findings from animal models to humans is fraught with many a broken promise, it is tempting to speculate on the potential implications for Alzheimer’s disease.

These results may offer a mechanistic explanation for the observations that cognitively normal individuals who are ApoE4 carriers show evidence for early neuronal dysfunction/synaptic failure (1,2). More recently, ApoE4 carriers were found to be especially susceptible to neurotoxic adverse effects observed in patients in a clinical trial of a humanized monoclonal antibody against amyloid-β. The spectrum of imaging abnormalities in these individuals includes vasogenic edema, sulcal effusions, microhemorrhages, and hemosiderin deposits (3). Whether or not the findings reported by Bell and colleagues will eventually lead to the identification of therapeutic targets against neuronal dysfunction or neurotoxicity in at-risk individuals remains to be seen.